Immunoassays or nucleic acid assays are carried out by a variety of means including visual and radioactivity analyses. Fluorescence intensities of fluorophores or substances labeled with fluorescent dyes are often measured because of the simplicity of the procedure. This technique is useful because the wavelength of the generated fluorescence or luminescence is in a range that is different from that of the excitation light, and can thus be accurately detected. Fluorescence or luminescence, however, disappears in a relatively short period of time, this enables the fluorescence or luminescence to be detected at substantially the same time as the generation of the excitation light, so that noises generated by the excitation light are also detected, and the background noises are sometimes heightened.
Peaks of the fluorescence or emission wavelength often exist in wavelength ranges that are longer than the peaks of the excitation wavelength. Fluorescence or emission is separated from the excitation light using, for example, a filter for selecting a wavelength and then its intensity is measured. The difference between the excitation wavelength and the emission or fluorescence wavelength is generally referred to as a Stokes shift. In fluorescence assays, a Stokes shift is small. Thus, it is sometimes difficult to distinguish the excitation light from the fluorescence emission based on differences in wavelengths.
Fluorescent dyes such as rhodamine and fluoresceine have wide fluorescence wavelength distributions, therefore broad fluorescence waveforms can be obtained. When simultaneous detections of several substances using several fluorophores or fluorescent dyes are intended, it is difficult to assay individual substances due to the overlapping fluorescence wavelength regions. Alternatively, this assay requires the application of a conversion formula to analyze the algorithm in order to find out the amount of each substance.
Chemiluminescence techniques including electrochemiluminescence technique and enzymatic chemiluminescence technique are sometimes employed as techniques in which excitation lights do not interfere with the emission light. In these techniques, labeled bodies or labeling dyes are not irradiated with the excitation light, and thus, the light emission only is detected. A technique has been developed in which isoluminol or acridium ester is labeled to detect the object substance. Another technique involves a highly-sensitive assay system that utilizes reactions between peroxidase and luminol in which the enzyme is employed as a labeling substance and this labeling substance allows a substrate to emit light, or reactions between adamantyl 1,2-dioxetane arylphosphate (AMPPD) and alkali phosphatase.
Fluorescence or phosphorescence of a complex comprising a rare earth element, such as europium or samarium, and a ligand thereof has a long lasting light emission. Recently, time-resolved fluorometry or phosphorimetry that makes use of this advantage has been developed as an excellent technique. Properties, such as a long period of fluorescence or phosphorescence being quenched, a large Stokes shift, and sharp peak waveforms of fluorescence and phosphorescence, enable the detection of fluorescent or phosphorescent signals while excluding noises caused by the excitation light. Thus, a highly sensitive analytical technique can be provided. In this technique, the excitation light is applied via a xenon flash lamp or laser beam while pulsing, and the fluorescence or phosphorescence of interest is detected after the fluorescence of the apparatus or other substances caused by the excitation lights disappears. This ligand is improved, and compounds such as 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) or 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl (BHHCT) have been reported. BHHCT is disclosed in, for example, JP-A-09-241233. These are excellent chelate compounds, although they are not yet improved enough to yield the minimal detection sensitivity required for immunoassays or nucleic acid assays.